Alternative pre-mRNA splicing greatly contributes to the mammalian proteomic diversity. The novel splice variants often emerge through splicing regulation at/nearby the splice sites (SS). A large group of 3′SS in human genes contain REPA (regulatory elements between the Py and 3′AG) G tracts that mostly appear in mammals as splicing silencers. However, the underlying molecular mechanisms and functional consequences remain unknown. We have identified a novel class of REPA G tracts (G)5-8 in a group of human genes including PRMT5 (protein arginine methyl transferase 5) that are significantly enriched in functional clusters of cell growth and proliferation. The PRMT5 G tracts emerged evolutionarily in mammals and repress splicing through recruitment of mainly hnRNP H that interferes with early spliceosome assembly. The splicing repression creates a shorter PRMT5 isoform (PRMT5S) that inhibits cell cycle progression contrary to the role of the full length protein (PRMT5L). Moreover, the expression of a group of genes involved in cell cycle arrest at interphase is preferentially regulated by PRMT5S. We further showed that PRMT5S is differentially expressed among cell and tissue types suggesting tissue-specific regulation. It exhibits distinct subcellular localization pattern from that of PRMT5L and opposite effects on cell cycle-specific structural dynamics of the Golgi apparatus. Moreover, these splice variants are differentially expressed during cell differentiation and PRMT5S promotes the differentiation of dendritic cells whereas PRMT5L shows the opposite effect. The expression of a large number of genes including those involved in crucial cellular processes such as differentiation and apoptosis is regulated by these splice variants of PRMT5. This study provides a direct link between the evolutionary emergence of a novel splicing regulatory G tract element and the generation of a functionally distinct protein isoform. The molecular mechanism underlying the splicing regulation by this G tract is likely common to many mammalian genes and the generation of their protein diversity.